Gregory L. Helms

The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms

The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the betaIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin- and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the betaIII isotype of tubulin was generated to evaluate the effect of betaIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the betaIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the betaIII-tubulin-overexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms. The ability of the taccalonolides to overcome clinically relevant mechanisms of drug resistance in vitro and in vivo confirms that the taccalonolides represent a valuable addition to the family of microtubule-stabilizing compounds with clinical potential.

(+)-Larreatricin hydroxylase, an enantio-specific polyphenol oxidase from the creosote bush (Larrea tridentata)

An enantio-specific polyphenol oxidase (PPO) was purified ≈1,700-fold to apparent homogeneity from the creosote bush (Larrea tridentata), and its encoding gene was cloned. The posttranslationally processed PPO (≈43 kDa) has a central role in the biosynthesis of the creosote bush 8–8′ linked lignans, whose representatives, such as nordihydroguaiaretic acid and its congeners, have potent antiviral, anticancer, and antioxidant properties. The PPO primarily engenders the enantio-specific conversion of (+)-larreatricin into (+)-3′-hydroxylarreatricin, with the regiochemistry of catalysis being unambiguously established by different NMR spectroscopic analyses; the corresponding (–)-enantiomer did not serve as a substrate. This enantio-specificity for a PPO, a representative of a widespread class of enzymes, provides additional insight into their actual physiological roles that hitherto have been difficult to determine.

Noble-metal catalyzed hydrodeoxygenation of biomass-derived lignin to aromatic hydrocarbons

Conversion of biomass derived lignin to liquid fuels has the promising potential to significantly improve carbon utilization and economic competitiveness of biomass refineries. In this study, an aqueous phase catalytic process was developed to selectively depolymerize the lignin polymeric framework and remove oxygen via hydrodeoxygenation (HDO) reactions. Efficient methods (ethanol and dilute alkali extraction) for selectively producing reactive lignin oligomers with high yields from corn stover were established. Characteristic structural features of the technical lignins employed for hydrocarbon production were elucidated with the aid of advanced analytical techniques, such as 2D HSQC NMR spectroscopy and gel permeation chromatography (GPC). Combinations of noble metal catalysts in the presence of various solid acid zeolites were tested for HDO activity of the oligomeric technical lignins predominantly containing 8–O–4′ inter-unit linkages. Results showed 35%–60% conversion of lignin with 65%–70% product selectivity for aromatic hydrocarbons (e.g. toluene) under various HDO conditions in the presence of noble metals (Ru, Rh and Pt) over Al2O3 (or C) supports and solid acid zeolites (e.g., NH4+ Z-Y 57277-14-1) catalyst matrices.

Antifungal compounds from idioblast cells isolated from avocado fruits

(E,Z,Z)-1-Acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene was isolated from avocado, Persea americana Mill., idioblast cells. It inhibited spore germination of the fungal pathogen Colletotrichum gloeosporioides. Full characterization is also reported for two additional compounds that have been described and partially characterized previously.

Identification and biological activities of new taccalonolide microtubule stabilizers.

The taccalonolides are a unique class of microtubule stabilizers that do not bind directly to tubulin. Three new taccalonolides, Z, AA, and AB, along with two known compounds, taccalonolides R and T, were isolated from Tacca chantrieri and Tacca integrifolia. Taccalonolide structures were determined by 1D and 2D NMR methods. The biological activities of the new taccalonolides, as well as taccalonolides A, B, E, N, R, and T, were evaluated. All nine taccalonolides display microtubule stabilizing activity, but profound differences in antiproliferative potencies were noted, with IC(50) values ranging from the low nanomolar range for taccalonolide AA (32 nM) to the low micromolar range for taccalonolide R (13 μM). These studies demonstrate that diverse taccalonolides possess microtubule stabilizing properties and that significant structure-activity relationships exist. In vivo antitumor evaluations of taccalonolides A, E, and N show that each of these molecules has in vivo antitumor activity.

Placement of 19F into the center of GB1: effects on structure and stability

A structural and thermodynamic characterization of 5F‐Trp‐substituted immunoglobulin binding domain B1 of streptococcal protein G (GB1) was carried out by nuclear magnetic resonance and circular dichroism spectroscopy. A single fluorine reporter atom was positioned at the center of the three‐dimensional structure, uniquely poised to be exploited for studying interior properties of this protein. We demonstrate that the introduction of 5F‐Trp does not affect the global and local architecture of GB1 and has no influence on the thermodynamic stability. The favorable properties of the fluorinated GB1 render this molecule a desirable model system for the development of spectroscopic methodology and theoretical calculations.

5-O-(α-D-galactopyranosyl)-D-glycero-pent-2-enono-1,4-lactone : Characterization in the oxalate-producing fungus, Sclerotinia sclerotiorum

Extracts of sclerotia from Sclerotinia sclerotiorum, a fungal phytopathogen, contain two electrochemically-active constituents, D-glycero-pent-2-enono-1,4-lactone (trivial name: D-erythroascorbic acid), and a previously unidentified compound, here characterized as 5-O-(alpha-D-galactopyranosyl)-D-glycero-pent-2-enono-1,4-lactone on the basis of its physical and chemical properties and its two hydrolytic products, D-galactose and D-erythroascorbic acid. Treatment of this galactoside with alkaline hydrogen peroxide produces oxalic acid as observed earlier with erythroascorbic acid.

Evelynin, a Cytotoxic Benzoquinone-Type retro-Dihydrochalcone from Tacca chantrieri

A new benzoquinone-type retro-dihydrochalcone, named evelynin, was isolated from the roots and rhizomes of Tacca chantrieri. The structure was elucidated on the basis of the analysis of spectroscopic data and confirmed by a simple one-step total synthesis. Evelynin exhibited cytotoxicity against four human cancer cell lines, MDA-MB-435 melanoma, MDA-MB-231 breast, PC-3 prostate, and HeLa cervical carcinoma cells, with IC(50) values of 4.1, 3.9, 4.7, and 6.3 μM, respectively.

Sequence‐Controlled Oligomers Fold into Nanosolenoids and Impart Unusual Optical Properties

Controlled syntheses give unique block oligomers with alternating flexible ethylene glycol and rigid perylenetetracarboxylic diimide (PDI) units. The number of rigid units vary from n=1 to 10. PDI units were stitched together by using efficient phosphoramidite chemistry. The resulting oligomers undergo folding in most solvents, including chloroform. In their ground state, these folded oligomers were characterized by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), NMR spectroscopy, and electronic absorption spectroscopy. FTICR-MS revealed the exact masses of these sequence-controlled oligomers, which confirmed the chemical composition and validated the synthetic strategy. The NMR neighboring ring-current effect (NRE) indicates the formation of cofacial π stacks; the stacked aromatic rings have nearly coaxial alignment akin to a nanosoleniod. Nanosolenoidal shielding in π stacks causes all aromatic protons to shift upfield, whereas NOE in a cyclic hetero-chromophoric dimer supports a rotated, cofacial π-stacking orientation separated by about 3.5 Å. Electron-phonon coupling is much stronger than excitonic coupling in these self-folded PDI oligomers; thus, Franck-Condon factors dictate the observed spectral features in visible spectra. The absorbance spectrum exhibits weak hypochromism due to π stacking with increasing stacking units n. Finally, ab initio calculations support the experimental observations, indicating 3.5 Å cofacial spacing in which one molecule is rotated 30° from the eclipsed orientation and higher oligomers can adopt, without a compensating energy penalty, either the right/left-handed helices or the 1,3-eclipsed structures. Both theory and experiments validate the nano-π-solenoids and their novel photophysical properties.

Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase.

To facilitate evaluation of enzyme–ligand complexes in solution, we have isolated the 26‐kDa N‐terminal domain of 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase for analysis by NMR spectroscopy. The isolated domain is capable of binding the substrate shikimate‐3‐phosphate (S3P), and this letter reports the localization of the S3P binding site using chemical shift mapping. Based on the NMR data, we propose that Ser23, Arg27, Ser197, and Tyr200 are directly involved in S3P binding. We also describe changes in the observed nuclear Overhauser effects (NOEs) that are consistent with a partial conformational change in the N‐terminal domain upon S3P binding.